Improvement of yeast biochip sensitivity using multilayer inorganic sol-gel substrates.

Today, microarray fluorescence detection is still limited because a great proportion of hybrids remain undetectable. In this paper we describe sol-gel optical multilayers (stacks of low- and high-index layers) deposited on glass slides which increase the fluorescence of DNA microarrays and favour the detection of fluorescent targets. An alternative to the expensive and time-consuming physical vapour deposition technology is proposed. It is a low-cost sol-gel coating of glass slides, each layer being made by "dipping" (alternatively in SiO2 or TiO2 solutions), "draining and drying". After the selection of the best surface layer of the substrates, the multilayer mirrors modelled for one (Cy3) or two (Cy3 and Cy5) fluorophores are spotted with a series of Yeast probes and compared to similar microarrays on standard glass slides through hybridisation experiments. The fluorescence images of the mirrors show increased signals for all the probes. The enhancement factors determined for Cy3 and for Cy3/Cy5 mirrors (10-12 and 4-5, respectively) are consistent with the initial modelling. This allows the assessment of the basal expression levels of Yeast low-expressed genes. Moreover, these substrates show a noticeable increase in sensitivity for induction/repression ratio measurements in differential gene expression experiments. So, they could be considered as promising tools for the analysis of small biological samples.

Multiple wavelength fluorescence enhancement on glass substrates for biochip and cell analyses.

In microarrays experiments, a serious limitation is the unreliability of low signal intensities data and the lack of reproducibility for the resulting ratios between samples and controls. Most of the light emitted by a fluorophore at the air/glass interface of a glass slide is absorbed by the glass so just a part of the emitted fluorescence is detected. To improve the sensitivity of the fluorescence detection of both common fluorophores Cy3 and Cy5 in DNA microarrays and fluorescent cell analyses, we have designed a multi layer mirror with alternative thin layers of SiO2 and HfO2. This mirror (MOTL) prevents fluorescence absorption, allows the simultaneous enhancement of the fluorescence signals and increases the dynamic range of the slides. Using MOTL slides, Cy3 and Cy5 intensities are enhanced by 5-8-fold, consequently, the fluorescence analysis becomes easier and should allow the detection of low copy number genes or weakly fluorescent cells. With the same approach, other multiple optical thin layer slides could be designed for other series of fluorophores, extending the field of their applications.

Direct in situ reverse transcriptase-linked polymerase chain reaction with biotinylated primers for the detection of hepatitis C virus RNA in liver biopsies.

To assess the presence and the cellular distribution of hepatitis C virus (HCV) RNA in the liver of 11 patients with confirmed HCV infection, a direct in situ reverse transcriptase-linked polymerase chain reaction (RT-PCR) method was performed on formalin-fixed and paraffin-embedded biopsies. The oligonucleotide primers used were specific to the 5' non coding region. An unlabelled downstream oligonucleotide served as a primer for reverse transcription as well as PCR. The upstream oligonucleotide serving as a primer for PCR was biotinylated, allowing a direct enzymatic detection of PCR products. HCV infected cells revealed cytoplasmic staining mainly concentrated towards the interface of the nucleus and cytoplasm. Most of the stained cells were hepatocytes and sometimes Kupffer cells. The results were compared with those obtained by RT-PCR of RNA extracted from the corresponding tissue block. Extracted HCV RNA could be detected in liver tissues of nine out of 11 (82%) infected patients. The detection rate using in situ RT-PCR was 7/11 (63%). The use of labelled primers improved specificity of direct in situ methods, by preventing non-specific incorporation of labelled dNTPs into fragmented DNA. Further studies are however required in order to increase detection sensitivity of HCV infection by in situ molecular methods.

Polypyrrole DNA chip on a silicon device: example of hepatitis C virus genotyping.

We describe in this article an oligonucleotide array constructed on a silicon device bearing a matrix of addressable 50-microns microelectrodes. Each electrode was covered by a conducting polymer (polypyrrole) grafted by an oligonucleotide (ODN). The DNA chip was prepared by successive electrochemically addressed copolymerizations of 5' pyrrole-labeled ODN and pyrrole. Following hybridization of the biotinylated amplified sample on the chip bearing a series of probes, detection was carried out by fluorescence microscopy through an R-phycoerythrin label. This technology was successfully applied to the genotyping of hepatitis C virus in blood samples. Results show good sensitivity and a high degree of dimensional resolution.

Rapid diagnosis of Mycobacterium tuberculosis infections by an ELISA-like detection of polymerase chain reaction products.

A polymerase chain reaction (PCR) assay was developed for the detection in clinical samples of mycobacteria belonging to the Mycobacterium tuberculosis complex. PCR products were detected with a simple and rapid colormetric method. With this method, 50 fg of M. tuberculosis DNA were detectable with the repetitive DNA-sequence-derived primers, corresponding to 10 genome equivalents. Detection of M. tuberculosis in 258 clinical samples by PCR was compared with detection by culture. PCR was positive for 56 of 57 culture-positive and Ziehl-Neelsen-staining-positive (ZN) samples, 11 of 18 culture-positive and ZN-negative samples. The presence of groEL DNA sequences was also investigated by PCR for all the specimens with the same revelation protocol. Three of the eight false-negative samples with the repetitive element-derived primers were found to contain groEL DNA sequences specific for the Mycobacterium genus. Among the 183 culture-negative samples, 30 were positive by PCR. When clinical data were known, the diagnosis of tuberculosis was established for the patients from whom those samples had been obtained. The results show that the rapid and simplified PCR assay described here is slightly more sensitive than culture and can be used in routine clinical practice.

Detection of HIV1 DNA in biological samples by an homogeneous assay: fluorescence measurement of double-stranded RNA synthesized from amplified DNA.

A nonisotopic homogeneous detection of nucleic acid sequences after amplification is described. We show that a DNA fragment bearing T7 RNA polymerase promoters on each extremity is able to be transcribed in two complementary RNAs, leading to a high yield direct synthesis of double-stranded RNA (dsRNA). Thus, this dsRNA can be easily detected and quantified in solution by fluorescence in the presence of propidium iodide. This reaction, used as a postamplification step, has been associated with a nested polymerase chain reaction (PCR); the second PCR round allowing the incorporation of the T7 promoters. This leads to a very efficient homogeneous assay. The fluorescence signal is proportional to the concentration of PCR product and is highly specific. This method can be easily carried out with currently available reagents and with unsophisticated instrumentation. This homogeneous procedure has been evaluated for the detection of HIV1 in blood samples; the sensitivity and the specificity appear to be equivalent to that of the radioactive method.

Comparison of three primer sets for the detection of Mycobacterium tuberculosis in clinical samples by polymerase chain reaction.

A number of studies have underlined the interest of the polymerase chain reaction (PCR) in the detection of Mycobacterium tuberculosis in clinical samples. Among the different parameters to be carefully studied the choice of target gene and primers is essential. The amplification of nucleotidic sequences localised on three different target genes (groEL, IS6110, Pab) was examined in 196 clinical samples from patients with suspected tuberculosis or receiving antituberculous therapy. The results obtained after hybridization with non-radioactive labelled probes were compared with the culture data. None of the primer sets studied showed a satisfactory sensitivity (79% to 84%) suitable for it to be used alone. The false-negative specimens with the PCR tests usually corresponded to those that contained few mycobacteria. With the methods described in this study, the use of two or three primer sets located on different target genes allowed to improve the positivity rate compared to the culture and sensitivity of the test (90-98%), particularly for paucibacillary samples. On the other hand, the interpretation was easier when concordant results were obtained.

Fast solid support detection of PCR amplified viral DNA sequences using radioiodinated or hapten labelled primers.

Oligonucleotides with novel modifications have been synthesized and incorporated into enzymatically amplified DNA sequences. They allow the fast detection of viral DNA sequences after two rounds of amplification. The hybrids formed are immobilized by affinity on coated tubes and detected by direct beta (32P) or gamma (125I) counting or by colorimetric revelation. The effect of a dilution step between the two amplifications is studied to obtain optimal sensitivity and specificity. This test is used to detect Human Papillomavirus types 16 and 18 in cells and biopsies and for the specific colorimetric detection of HIV1 in extracted DNA.

Histochemical detection of the messenger RNAs coding for calcitonin and calcitonin gene-related peptide in medullary thyroid carcinomas with radioactive and biotinylated oligonucleotide probes.

The present study has been undertaken to investigate the efficiency of biotinylated synthetic oligonucleotide probes in detection by in situ hybridization of the mRNAs coding for calcitonin (CT) or calcitonin gene-related peptide (CGRP) in human medullary thyroid carcinomas (MTCs). Tissue sections fixed with formaldehyde were hybridized with 45-base long oligonucleotides, specific for CT or CGRP mRNA. Recombinant DNA probe or synthetic oligonucleotides radioactively labelled with 32P or 35S were used as controls to detect by autoradiography the corresponding mRNAs in the tumour cells. Oligonucleotide probes labelled by fixation of one biotin molecule at their 5'-end, or by incorporation of a tail of biotin-11-dUTP at their 3'-end, were used and were revealed by incubation with streptavidin-alkaline phosphatase associated with the corresponding substrate. Each biotinylated probe stained exclusively the cytoplasm of the tumour cells, the CT probe giving a much higher level of staining than the CGRP probe. The same cells were found to contain CT and CGRP mRNAs. Controls performed with either radioactive or biotinylated probes confirmed the specificity of the staining. These results demonstrate that biotinylated synthetic oligonucleotides can be used as efficient tools to investigate gene expression in tissue sections, thus avoiding the various inconveniences connected with the use of radioactive probes, especially bio-hazards, the use of autoradiography, the limited histological resolution, and the delay in obtaining results.

Dopamine receptor gene expression by enkephalin neurons in rat forebrain.

In situ hybridization experiments were performed with brain sections from normal, control and haloperidol-treated rats to identify and map the cells expressing the D2 dopamine receptor gene. D2 receptor mRNA was detected with radioactive or biotinylated oligonucleotide probes. D2 receptor mRNA was present in glandular cells of the pituitary intermediate lobe and in neurons of the substantia nigra, ventral tegmental area, and forebrain, especially in caudate putamen, nucleus accumbens, olfactory tubercle, and piriform cortex. Hybridization with D2 and preproenkephalin A probes in adjacent sections, as well as combined hybridization with the two probes in the same sections, demonstrated that all detectable enkephalin neurons in the striatum contained the D2 receptor mRNA. Large neurons in caudate putamen, which were unlabeled with the preproenkephalin A probe and which may have been cholinergic, also expressed the D2 receptor gene. Haloperidol treatment (14 or 21 days) provoked an increase in mRNA content for D2 receptor and preproenkephalin A in the striatum. This suggests that the increase in D2 receptor number observed after haloperidol treatment is due to increased activity of the D2 gene. These results indicate that in the striatum, the enkephalin neurons are direct targets for dopamine liberated from mesostriatal neurons.

Vasopressin gene expression in the normal and Brattleboro rat: a histological analysis in semi-thin sections with biotinylated oligonucleotide probes.

We analyzed expression of the vasopressin (AVP) gene in semi-thin sections in normal and Brattleboro rats by using in situ hybridization and immunohistochemistry. AVP mRNA was detected as follows: vibratome sections of rat hypothalamus were hybridized with a biotinylated oligonucleotide probe, embedded in Araldite, and cut into semi-thin sections which were reacted with streptavidin-alkaline phosphatase and the appropriate substrate. Adjacent serial sections were treated by immunohistochemistry to detect AVP or oxytocin immunoreactivity. In normal rat, AVP mRNA can be detected in magnocellular neurons of the supraoptic and paraventricular nuclei and in parvocellular neurons of the suprachiasmatic nucleus. AVP mRNA was present throughout the cytoplasm of the cell bodies, their processes, and in punctate structures in the vicinity of the AVP cell bodies. Most neurons containing AVP mRNA also contain AVP immunoreactivity, but the staining intensity was not consistently correlated for each reaction. A few neurons contained AVP mRNA without detectable AVP immunoreactivity. In the Brattleboro rat, staining intensity of the reaction was lower than in normal rat and the AVP mRNA was restricted mostly to the periphery of the cytoplasm. In this strain, the neurons containing the AVP mRNA did not contain AVP or oxytocin immunoreactivity. These results demonstrate that neuropeptide mRNA can be detected in semi-thin sections with a biotinylated oligonucleotide probe, and that AVP gene deletion provokes modification of the intracellular localization of the AVP mRNA.

Histological detection of messenger RNAs with biotinylated synthetic oligonucleotide probes.

We achieved histological detection of the messenger RNAs coding for vasopressin, calcitonin, or calcitonin gene-related peptide by using biotinylated synthetic oligonucleotides, and defined the technical parameters enabling optimal detection of these mRNAs. Oligonucleotides labeled by fixation of one biotin at their 5' end or by addition of a biotin-11-dUTP tail at their 3' end can be used to detect mRNAs, although the latter are more sensitive. Streptavidin-alkaline phosphatase revealed with nitroblue tetrazolium-bromo-chloro-indolyl phosphate as substrate makes possible detection of the biotinylated oligonucleotides. Increasing formaldehyde concentration in the fixative decreases the signal intensity; 1% formaldehyde fixation provides the most intense signal. Several controls, including those with addition of unlabeled oligonucleotides to the hybridization buffer, confirm the specificity of mRNA detection. The sensitivity of the biotinylated probes is identical or lower as compared to the corresponding radiolabeled oligonucleotides. Histological and subcellular resolution is greatly enhanced with biotinylated probes. The rat vasopressin probes stain magnocellular neurons in the supraoptic and paraventricular nuclei and, under optimal conditions, parvocellular neurons in the suprachiasmatic nucleus. Vasopressin mRNA is present in the cytoplasm of the cell bodies and in the roots of certain processes. Calcitonin and calcitonin gene-related peptide mRNA are found co-localized in the cytoplasm of the same tumor cells in human medullary thyroid carcinoma.

Synthesis and spectroscopic properties of two classes of 5,6-dihydrothymidine derivatives. Action on the Ehrlich's ascites cells thymidine kinase.

Trans (+) and (-) 6-alkoxy-5-bromo-5,6 dihydrothymidine and trans (+) and (-) 6-alkoyloxy-5-bromo-5,6-dihydrothymidine compounds have been prepared. The synthesis of these substances (alkoxy : methoxy, ethoxy, butyloxy and isoamyloxy and alkoyloxy : acetoxy and bensovloxy) is described. Diastereoisomers of all products have been isolated by thin layer chromatography and their spectroscopic properties (IR, UV, NMR, mass spectrometry) studied. These compounds have been shown to be competitive inhibitors of Ehrlich's ascites cells thymidine kinase with respect ot thymidine.